The primary objective of this research proposal is to elucidate the complete three-dimensional structure of the NAD-malic enzyme from the parasitic nematode, Ascaris suum. Attendant to this structure solution will be the solution of the mammalian malic enzyme. The elucidation of the structure of malic enzyme will be done by X-ray crystallography, circular dichroism, and fluorescence. Crystals of the ascarid enzyme have been obtained which diffract to 2.5 Angstrom units and we have obtained several native data sets at 2.5-3.0 Angstrom units. The next step is to make heavy metal derivatives, one has been made with uranyl acetate. The remainder will entail collecting at least two or three heavy metal derivatives, determining the phases, preparing an interpretable electron density map and solving the structure of the malic enzyme. Utilizing the structure obtained from X-ray studies we will carry out a comparative structure-based drug design study utilizing the computer program DOCK and various inhibitors of the malic enzyme. This study will also involve working with the structure of the mammalian malic enzyme. We will also carry out a study on the structure of malic enzyme by the techniques of circular dichroism and fluorescence. By studying the change in structure brought about by effectors, these changes can be correlated with the X-ray structure solution. A site- directed mutagenesis study will also be carried out using the clone for the malic enzyme and the expression system which has been developed in this laboratory. The purpose of this study is two-fold. First, to identify amino acids that are involved in the catalytic mechanism, and second, to characterize amino acids that bind to inhibitors identified in the drug design studies. The Altered Site in vitro mutagenesis system will be used to generate the mutant enzyme. Specific mutants of amino acids will be generated in the malate binding domain to determine the role of various residues in the catalytic sequence. Other mutants will be made of amino acids that are in the subunit interaction domain in order to ascertain if the enzyme can function as a monomer. Finally, mutants will be made in those amino acids identified by the drug design studies which bind inhibitors in order to further understand binding. These mutant enzymes will also be studied by circular dichroism and fluorescence to determine if any changes have occurred in the structure that can be detected by the methods. In addition, crystals will be made of the mutant enzymes and their structures can be solved by molecular replacement.